Summary: Clostridial neurotoxins (botulinum and tetanus toxins) are large proteins organized into three functional domains, an amino terminal proteolytic domain, central translocation domain, and a carboxyl terminal receptor binding region. The neurotoxic effects of clostridial neurotoxins results from binding to cell surface receptors, translocation into the neural cell, and the proteolytic cleavage of proteins essential for synaptic vesicle docking/fusion events (SNARE proteins) by the enzymatically active amino terminal domain. The subsequent block of neurotransmitter release at the neuromuscular junction by botulinum toxins or block of inhibitory neurotransmitter release within the central nervous system by tetanus toxin leads to flaccid or spasmodic paralysis of the victim, respectively. The focus of this project has been the the characterization of the receptor binding domain of tetanus C-fragment. To determine which amino acids in tetanus toxin are involved in ganglioside binding, homology modeling was performed using recently resolved X-ray crystallographic structures of tetanus toxin HC fragment. Based on these analyses, two regions in tetanus toxin that share structural homology with the binding domains of other sialic acid and galactose-binding proteins were targeted for mutagenesis. Specific amino acids within these regions were altered using site-directed mutagenesis. The amino acid residue tryptophan-1288 was found to be critical for binding of the HC fragment to ganglioside GT1b. Docking of GD1b within this region of the toxin suggested that histidine-1270 and aspartate-1221 were within hydrogen bonding distance of the ganglioside. These two residues were mutagenized and found to also be important for the binding of tetanus toxin HC fragment to ganglioside GT1b. In addition, the HC fragments mutagenized at these residues have reduced levels of binding to neurites of differentiated PC-12 cells. These studies indicate that the amino acids tryptophan-1288, histidine-1270, and aspartate-1221 are components of the GT1b-binding site on the tetanus toxin HC fragment.